Field of the Invention
The present invention is directed to multimeric hemoglobin-like proteins composed of two or more pseudotetramers linked together either by genetic fusion or by chemical crosslinking.
Description of the Background Art
A. Structure and Function of Hemoglobin PA0 B. Blood Substitutes, Generally PA0 C. Naturally Occurring Cysteine Substitution Mutants of Hemoglobin (Non-Polymerizing) PA0 D. Naturally Occurring Polymerizing or Polymeric Hemoglobins PA0 E. Artificially Crosslinked Hemoglobins (Non-Polymerizing) PA0 F. Artificially Crosslinked Hemoglobin (Polymerizing) PA0 G. Fused Genes and Proteins, Generally
Hemoglobin (Hgb) is the oxygen-carrying component of blood. Hemoglobin circulates through the bloodstream inside small enucleate cells called erythrocytes (red blood cells). Hemoglobin is a protein constructed from four associated polypeptide chains, and bearing prosthetic groups known as hemes. The erythrocyte helps maintain hemoglobin in its reduced, functional form. The heme iron atom is susceptible to oxidation, but may be reduced again by one of two enzyme systems within the erythrocyte, the cytochrome b.sub.5 and glutathione reduction systems.
The structure of hemoglobin is well known. We herewith incorporate by reference the entire text of Bunn and Forget, eds., Hemoglobin: Molecular, Genetic and Clinical Aspects (W. B. Saunders Co., Philadelphia, Pa.: 1986) and of Fermi and Perutz "Hemoglobin and Myoglobin," in Phillips and Richards, Atlas of Molecular Structures in Biology (Clarendon Press: 1981).
About 92% of the normal adult human hemolysate is Hgb A (designated alpha2 beta2, because it comprises two alpha and two beta chains). Other recognized hemoglobin species are Hgb A.sub.2 (.alpha..sub.2 .delta..sub.2), Hgb A.sub.1a, Hgb A.sub.1b, and Hgb A.sub.1c, as well as the rare species Hgb F (.alpha..sub.2 gamma.sub.2), Hgb Gower-1 (Zeta.sub.2 epsilon.sub.2) , Hgb Gower-2 (alpha.sub.2 epsilon.sub.2) , Hgb Portland (Zeta.sub.2 gamma.sub.2), and Hgb H (beta.sub.4) and Hgb Bart (gamma.sub.4). They are distinguished from Hgb A by a different selection of polypeptide chains.
The primary structure of a polypeptide is defined by its amino acid sequence and by identification of any modifications of the side chains of the individual amino acids. The amino acid sequences of both the alpha and beta globin polypeptide chains of "normal" human hemoglobin is given in Table 1. Many mutant forms are also known; several mutants are identified in Table 400. The wild-type alpha chain consists of 141 amino acids. The iron atom of the heme (ferroprotoporphyrin IX) group is bound covalently to the imidazole of His 87 (the "proximal histidine"). The wild-type beta chain is 146 residues long and heme is bound to it at His 92. Apohemoglobin is the heme-free analogue of hemoglobin; it exists predominantly as the .alpha..beta.-globin dimer.
Segments of polypeptide chains may be stabilized by folding into one of two common conformations, the alpha helix and the beta pleated sheet. In its native state, about 75% of the hemoglobin molecule is alpha-helical. Alpha-helical segments are separated by segments wherein the chain is less constrained. It is conventional to identify the alpha-helical segments of each chain by letters, e.g., the proximal histidine of the alpha chain is F8 (residue 8 of helix F). The non-helical segments are identified by letter pairs, indicating which helical segments they connect. Thus, nonhelical segment BC lies between helix B and helix C. In comparing two variants of a particular hemoglobin chain, it may be enlightening to attempt to align the helical segments when seeking to find structural homologies. For the amino acid sequence and helical residue notation for normal human hemoglobin A.sub.o alpha and beta chains, see Bunn and Forget, supra, and Table 1 herein.
The tertiary structure of the hemoglobin molecule refers to the steric relationships of amino acid residues that are far apart in the linear sequence, while quaternary structure refers to the way in which the subunits (chains) are packed together. The tertiary and quaternary structure of the hemoglobin molecule have been discerned by X-ray diffraction analysis of hemoglobin crystals, which allows one to calculate the three-dimensional positions of the very atoms of the molecule.
In its unoxygenated ("deoxy", or "T" for "tense") form, the subunits of hemoglobin A (alpha1, alpha2, beta1, and beta2) form a tetrahedron having a twofold axis of symmetry. The axis runs down a water-filled "central cavity". The subunits interact with one another by means of Van der Waals forces, hydrogen bonds and by ionic interactions (or "salt bridges"). The alpha1beta1 and alpha2beta2 interfaces remain relatively fixed during oxygenation. In contrast, there is considerable flux at the alpha1beta2 (and alpha2beta1) interface. In its oxygenated ("oxy", or "R" for "relaxed" form), the intersubunit distances are increased.
The tertiary and quaternary structures of native oxyhemoglobin and deoxyhemoglobin are sufficiently well known that almost all of the nonhydrogen atoms can be positioned with an accuracy of 0.5 .ANG. or better. For human deoxyhemoglobin, see Fermi, et al., J. Mol. Biol., 175: 159 (1984), and for human oxyhemoglobin, see Shaanan, J. Mol. Biol., 171: 31 (1983), both incorporated by reference.
Normal hemoglobin has cysteines at beta 93 (F9), beta 112 (G14), and alpha 104 (G11). The latter two positions are deeply buried in both the oxy and deoxy states; they lie near the .alpha..sub.1 .beta..sub.1 interface. Beta 93, however, in the oxy form is reactive with sulfhydryl reagents.
Native human hemoglobin has been fully reconstituted from separated heme-free alpha and beta globin and from hemin. Preferably, heme is first added to the alpha-globin subunit. The heme-bound alpha globin is then complexed to the heme-free beta subunit. Finally, heme is added to the half-filled globin dimer, and tetrameric hemoglobin is obtained. Yip, et al., PNAS (USA), 74: 64-68 (1977).
The human alpha and beta globin genes reside on chromosomes 16 and 11, respectively. Bunn and Forget, infra at 172. Both genes have been cloned and sequenced, Liebhaber, et al., PNAS 77: 7054-58 (1980) (alpha-globin genomic DNA); Marotta, et al., J. Biol. Chem., 252: 5040-51 (1977) (beta globin cDNA); Lawn, et al., Cell, 21:647 (1980) (beta globin genomic DNA).
Hemoglobin exhibits cooperative binding of oxygen by the four subunits of the hemoglobin molecule (two alpha-globins and two beta-globins in the case of Hgb A), and this cooperativity greatly facilitates efficient oxygen transport. Cooperativity, achieved by the so-called heme-heme interaction, allows hemoglobin to vary its affinity for oxygen. Hemoglobin reversibly binds up to four moles of oxygen per mole of Hgb.
Oxygen-carrying compounds are frequently compared by means of a device known as an oxygen dissociation curve. This curve is obtained when, for a given oxygen carrier, oxygen saturation or content is graphed against the partial pressure of oxygen. For Hgb, the percentage of saturation increases with partial pressure according to a sigmoid relationship. The P.sub.50 is the partial pressure at which the oxygen-carrying solution is half saturated with oxygen. It is thus a measure of oxygen-binding affinity; the higher the P.sub.50, the more loosely the oxygen is held.
When the oxygen dissociation curve of an oxygen-carrying solution is such that the P.sub.50 is less than that for whole blood, it is said to be "left-shifted."
The oxygen affinity of hemoglobin is lowered by the presence of 2,3-diphosphoglycerate (2,3-DPG), chloride ions and hydrogen ions. Respiring tissue releases carbon dioxide into the blood and lowers its pH (i.e. increases the hydrogen ion concentration), thereby causing oxygen to dissociate from hemoglobin and allowing it to diffuse into individual cells.
The ability of hemoglobin to alter its oxygen affinity, increasing the efficiency of oxygen transport around the body, is dependent on the presence of the metabolite 2,3-DPG. Inside the erythrocyte 2,3-DPG is present at a concentration nearly as great as that of hemoglobin itself. In the absence of 2,3-DPG "conventional" hemoglobin binds oxygen very tightly and would release little oxygen to respiring tissue.
Aging erythrocytes release small amounts of free hemoglobin into the blood plasma where it is rapidly bound by the scavenging protein haptoglobin. The hemoglobin-haptoglobin complex is removed from the blood and degraded by the spleen and liver.
Isolated alpha globin chains are monomers; exhibit high oxygen affinity but of course lack subunit cooperativity. Isolated beta globin chains aggregate to form a .beta..sub.4 tetramer (HbH). The .beta..sub.4 tetramer has a high but noncooperative oxygen affinity.
It is not always practical to transfuse a patient with donated blood. In these situations, use of a red blood cell substitute is desirable. The product must effectively transport O.sub.2, just as do red blood cells. ("Plasma expanders", such as dextran and albumin, do not transport oxygen.) The two types of substitutes that have been studied most extensively are hemoglobin solutions and fluorocarbon emulsions.
It is clear from the above considerations that free native hemoglobin A, injected directly into the bloodstream, would not support efficient oxygen transport about the body. The essential allosteric regulator 2,3-DPG is not present in sufficient concentration in the plasma to allow hemoglobin to release much oxygen at venous oxygen tension.
Nonetheless, solutions of conventional hemoglobin have been used as RBC substitutes. The classic method of preparing hemoglobin solutions employs outdated blood. The red cells are lysed and cellular debris is removed, leaving what is hopefully "stromal-free hemoglobin" (SFH).
Several basic problems have been observed with this approach. The solution must be freed of any toxic components of the red cell membrane without resorting to cumbersome and tedious procedures which would discourage large-scale production. DeVenuto, "Appraisal of Hemoglobin Solution as a Blood Substitute", Surgery, Gynecology and Obstetrics, 149: 417-436 (1979).
Second, as expected, such solutions are "left-shifted" (lower P.sub.50) as compared to whole blood. Gould, et al., "The Development of Polymerized Pyridoxylated Hemoglobin Solution as a Red Cell Substitute", Ann. Emerg. Med. 15: 1416-1419 (Dec. 3, 1986). As a result, the oxygen affinity is too high to unload enough oxygen into the tissues. Benesch and Benesch, Biochem. Biophys. Res. Comm., 26:162-167 (1967).
Third, SFH has only a limited half-life in the circulatory system. This is because oxy Hgb partially dissociates into a dimer (.alpha..beta.) that is rapidly cleared from the blood by glomerular filtration and binding to circulating haptoglobulin. If large amounts of soluble hemoglobin are introduced into the circulation, glomerular filtration of the dimers may lead to a protein and iron load on the kidney capable of causing renal damage. Bunn, H. F., et al. (1969) The renal handling of hemoglobin I. Glomerular filtration. J. Exp. Med. 129:909-923; Bunn, H. F., and J. H. Jandl; (1969) The renal handling of hemoglobin II. Catabolism. J. Exp. Med. 129:925-934; Lee, R. L., et al. (1989) Ultrapure, stroma-free, polymerized bovine hemoglobin solution: Evaluation of renal toxicity (blood substitutes) J. Surgical Res. 47:407-411; Feola, M., et al. (1990) Nephrotoxicity of hemoglobin solutions. Biomat. Art. Cell Art. Org., 18(2):233-249; Tam, S. C. and J. T. F. Wong (1988) Impairment of renal function by stroma-free hemoglobin in rats. J. Lab. Clin. Med. 111:189-193.
Finally, SFH has a high colloid osmotic pressure (COD). Thus, administration of SFH in a dose that would have the same oxygen-carrying capacity as a unit of packed red blood cells is inadvisable, since the high osmotic pressure (60 mm Hg) would cause a massive influx of water from the cells into the bloodstream, thus dehydrating the patient's tissues. This consideration limits the dose of SFH to that which provide a final concentration of about 6-8 gm Hgb/dl.
In an effort to restore the desired P.sub.50, researchers added 2,3-DPG to the hemoglobin solution. Unfortunately, 2,3-DPG was rapidly eliminated from the circulation. Scientists then turned to other organic phosphates, particularly pyridoxal phosphate. Like 2,3-DPG, these compounds stabilized the "T state" of the Hgb by forming a salt bridge between the N-termini of the two beta chains. The pyridoxylated hemoglobin had a P.sub.50 of 20-22 torr, as compared to 10 torr for SFH and 28 torr for whole blood. While this is an improvement over SFH, the pyridoxylated Hgb remains "high affinity" relative to whole blood.
There are a few known naturally occurring mutants of human hemoglobin in which a cysteine residue is substituted for another residue of normal hemoglobin Ao.
In hemoglobin Nigeria, the mutation is .alpha. 81 Ser.fwdarw.Cys; no disulfide is formed. Horis, et al., Blood, 55(1):131-137 (1980). In Hemoglobin Rainier, an intrasubunit disulfide is formed between the wild type F9(93).beta. Cysteine and the cysteine introduced by replacement of the Tyr at HC2(145).beta.. Greer, et al., Nature [New Biology], 230:261-264 (1971). Hemoglobin Nunobiki (.beta. 141 Drg.fwdarw.Cys) also features a non-polymerizing cysteine substitution. In both Hb Rainier and Hb Nunobiki, the new cysteine residues are on the surface of the tetramer.
Three other human mutants are known which polymerize as a result of formation of intermolecular (first tetramer to second tetramer) disulfide bridges. In Hemoglobin Porto Alegre, the Ser at A6(9).beta. is replaced by Cysteine, and since this cysteinyl residue is externally oriented, spontaneous polymerization occurs, and results in formation of a dodecamer with three Porto Alegre tetramers linked by disulfide bonds. An octamer has also been made by a 1:1 mixture of Porto Alegre hemoglobin and normal hemoglobin. Tondo, Biochem. Biophys. Acta, 342:15-20 (1974); Tondo, An. Acad. Bras. Ci, 59:243-251 (1987).
Hb Mississippi is characterized by a cysteine substitution in place of Ser CD3(44).beta.. Hemolysates of a patient were subjected to gel filtration column chromatography, and 48.8% eluted in the void volume. Since the molecular weight exclusion was about 600 kD, this suggested that Hb MS polymers are composed of ten or more hemoglobin tetramers. Adams, et al., Hemoglobin, 11(5):435-452 (1987).
A .beta.83(EF7)Gly.fwdarw.Cys mutation characterizes Hemoglobin Ta Li. This mutant showed slow mobility in starch gel electrophoresis, indicating that it was a polymer.
Polymeric mouse hemoglobins have been reported. In BALB/cJ mice, there is a reactive cysteinyl residue near the NH.sub.2 -terminal of the beta chain (.beta.-13 in the mouse). This mouse mutant has been compared to Hemoglobin Porto Alegre, which likewise has a cysteinyl residue in the A-helix of the beta chain. Octamer formation is most common. However, each tetramer has two extra cysteinyl residues, one on each .beta.-chain, that may react with different tetramers; "this explains why aggregates larger than octamers occur". Bonaventura and Riggs, Science, 158:800-802 (1967); Riggs, Science, 147:621-623 (1965).
Macaques also exhibit a polymerizing hemoglobin variant. Takenaka, et al., Biochem. Biophys. Acta, 492:433-444 (1977); Ishimoto, et al., J. Anthrop. Soc. Nippon, 83(3):233-243 (1975). This mutant has been compared to the Ta Li variant in humans.
Both amphibians and reptiles possess polymerizing hemoglobins. For example, in the bullfrog, hemoglobin "Component C" polymerizes by disulfide bond formation between tetramers. This is said to be primarily dependent on cysteinyl residues of the alpha chain. Tam, et al., J. Biol. Chem., 261:8290-94 (1986).
The extracellular hemoglobin of the earthworm (Lumbricus terrestris) has a complex structure. There are twelve subunits, each being a dimer of structure (abcd): where "a", "b", "c", and "d" denote the major heme containing chains. The "a", "b", and "c" chains form a disulfide-linked trimer. The whole molecule is composed of 192 heme-containing chains and 12 non-heme chains, and has a molecular weight of 3800 kDa. Other invertebrate hemoglobins are also large multi-subunit proteins.
The brine shrimp Artemia produces three polymeric hemoglobins with nine genetically fused globin subunits. Manning, et al., Nature, 348:653 (1990). These are formed by variable association of two different subunit types, a and b.beta.. Of the eight intersubunit linkers, six are 12 residues long, one is 11 residues and one is 14 residues.
The properties of hemoglobin have been altered by specifically chemically crosslinking the alpha chains between the Lys99 of alpha1 and the Lys99 of alpha2. Walder, U.S. Pat. No. 4,600,531 and 4,598,064; Snyder, et al., PNAS (USA) 84: 7280-84 (1987); Chaterjee, et al., J. Biol. Chem., 261:9929-37 (1986). The beta chains have also been chemically crosslinked. Kavanaugh, et al., Biochemistry, 27: 1804-8 (1988). Kavanaugh notes that the beta N-termini are 16 .ANG. apart in the T state and 20 .ANG. apart in the R state. Not surprisingly, the introduction of a DIDS bridge between the N-termini of T state hemoglobin hindered the shift to the R state, thereby decreasing the O.sub.2 affinity of the molecule. While the Kavanaugh analogue has desirable oxygen binding and renal clearance characteristics, it too is obtained in low yield.
Hoffman and Nagai, U.S. Pat. No. 5,028,588 suggest that the T state of hemoglobin may be stabilized by intersubunit (but intratetrameric) disulfide crosslinks resulting from substitution of cysteine residues for other residues. A particularly preferred crosslink was one connecting beta Gly Cys with either alpha G17 (Ala.fwdarw.Cys) or G18 (Ala.fwdarw.Cys).
Bonsen, U.S. Pat. No. 4,001,401, U.S. Pat. No. 4,001,200, and U.S. Pat. No. 4,053,590 all relate to polymerization of red blood cell-derived hemoglobin by chemical crosslinking. The crosslinking is achieved with the aid of bifunctional or polyfunctional crosslinking agents, especially those reactive with exposed amino groups of the globin chains. The result of the crosslinking reaction is a polydisperse composition of covalently cross-linked aggregates.
Bonhard, U.S. Pat No. 4,336,248 discloses chemical crosslinking of hemoglobin molecules to each other, or to serum proteins such as albumin.
Bonhard, U.S. Pat. No. 4,777,244 sought to stabilize the dialdehyde-cross-linked hemoglobins of the prior art, which tended to polymerized further while in storage, by adding a reducing agent to stabilize the azomethine bond.
Bucci, U.S. Pat. No. 4,584,130, at col. 2, comments that "the polyhemoglobin reaction products are a heterogeneous mixture of various molecular species which differ in size and shape. The molecular weights thereof range from 64,500 to 600,000 Daltons. The separation of individual molecular species from the heterogeneous mixture is virtually impossible. In addition, although longer retention times in vivo are obtained using polyhemoglobins, the oxygen affinity thereof is higher than that of stroma-free hemoglobin."
According to Tye, U.S. Pat. No. 4,529,719, "most workers have chosen to form the random intermolecular crosslinked polymers of hemoglobin because they believed that the 65,000 Dalton tetramer was filtered by the glomerulus . . . . Usually the amino groups of lysine on the surface of the hemoglobin molecule are coupled with a bifunctional reactant such as gluteraldehyde or suberimidate. There are 42 lysines available for reaction per hemoglobin tetramer so that one can get an infinite number of different inter [or] intra molecular crosslinks making various polymers of hemoglobin . . . . The random polymerization is difficult to control and gives a range between two and ten tetramers per polymer . . . . No one has yet standardized an analytical scheme to establish lot to lot variability of structure and function . . . . [Polymerized pyridoxylated hemoglobin has] a profound chemical heterogeneity making it difficult to study as a pharmaceutical agent."
Genes may be fused together by removing the stop codon of the first gene, and joining it in phase to the second gene. Parts of genes may also be fused, and spacer DNAs which maintain phase may be interposed between the fused sequences. The product of a fused gene is a single polypeptide, not a plurality of polypeptides as is expressed by a polycistronic operon. Different genes have been fused together for a variety of purposes. Thus, Gilbert, U.S. Pat. No. 4,338,397 inserted a rat preproinsulin gene behind a fragment of the E. coli penicillinase gene. His purpose was to direct E. coli transformants to secrete the expression product of the fused gene. Fused genes have also been prepared so that a non-antigenic polypeptide may be expressed already conjugated to an immunogenic carrier protein.
The use of linker DNA sequences to join two different DNA sequences is known. These linkers are used to provide restriction sites for DNA cleavage, or to encode peptides having a unique character that facilitates purification of the encoded fusion protein or a fragment thereof. See, e.g., Rutter, U.S. Pat. No. 4,769,326.
Hallewell, et al., J. Biol. Chem., 264: 5260-68 (1989) prepared an analogue of CuZn superoxide dismutase. Each dismutase molecule is a dimer of two identical subunits; a copper ion and a zinc ion are liganded to the subunit. The dimer interaction in CuZn superoxide dismutase is so strong that the subunits have not been separated without inactivating the enzyme. The enzyme has considerable conformational similarity to immunoglobulins; Hallewell, et al., joined two human superoxide dismutase genes, either directly or with DNA encoding a 19-residue human immunologlobulin IgA1 hinge region and expressed the fused genes in a transformed host. In attempting to express the directly joined genes, recombination occurred to eliminate one of the tandem genes in some plasmid molecules. Hallewell, et al., postulated that the direct connection distorted the dimer, causing the exposure of hydrophobic areas which then had a toxic effect. This would have provided selection pressure favoring gene deletion. No recombination was detected with the IgA1 linker construction.
Hoffman, et al., WO88/09179 describe the production, in bacteria and yeast, of hemoglobin and analogues thereof. The disclosed analogues including hemoglobin proteins in which one of the component polypeptide chains consists of two alpha or two beta globin amino acid sequences covalently connected by peptide bonds, preferably through an intermediate linker of one or more amino acids, without branching. In normal hemoglobin, the alpha and beta globin subunits are non-covalently bound.